Targeted Vectors

ABSTRACT

This invention provides therapeutic and diagnostic agent delivery vehicles, including viral vectors, that are complexed to a targeting moiety by coordinate covalent linkages mediated by a transition metal ion. The complex is typically formed with a transition metal ion that is in a kinetically labile oxidation state; after the complex is formed, the oxidation state of the transition metal ion is changed to one that renders the complex kinetically stable. The use of a coordinate covalent linkage to attach the targeting moiety to the delivery vehicle provides advantages such as the ability to readily attach a different targeting moiety to a delivery vehicle without modifying the delivery vehicle itself. This flexibility is achieved without sacrificing stability of the complex.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No.60/159,782, filed Oct. 15, 1999, which application is incorporated byreference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of targeting of gene deliverysystems (viral and non-viral) to particular cell and tissue types.

2. Background

The use of recombinant viral vectors for the delivery of exogenous genesto mammalian cells is well established. See e.g. Boulikas, T. in GeneTherapy and Molecular Biology Volume 1, pages 1-172 (Boulikas, Ed.)1998, Gene Therapy Press, Palo Alto, Calif. However, certain viralvectors commonly used in such instances, such as adenoviruses, exhibit abroad tropism which permits infection and expression of the exogenousgene in a variety of cell types. While this can be useful in someinstances, the treatment of certain diseases is enhanced if the virus isable to be modified so as “target” (i.e., to preferentially infect) onlya limited type of cell or tissue.

A variety of approaches to create targeted viruses have been describedin the literature. For example, cell targeting has been achieved withadenovirus vectors by selective modification of the viral genome knoband fiber coding sequences to achieve expression of modified knob andfiber domains having specific interaction with unique cell surfacereceptors. Examples of such modifications are described in Wickham etal. (1997) J. Virol. 71(11):8221-8229 (incorporation of RGD peptidesinto adenoviral fiber proteins); Arnberg et al. (1997) Virology227:239-244 (modification of adenoviral fiber genes to achieve tropismto the eye and genital tract); Harris and Lemoine (1996) TIG12(10):400-405; Stevenson et al. (1997) J. Virol. 71(6):4782-4790;Michael et al. (1995) Gene Therapy 2:660-668 (incorporation of gastrinreleasing peptide fragment into adenovirus fiber protein); and Ohno etal. (1997) Nature Biotechnology 15:763-767 (incorporation of ProteinA-IgG binding domain into Sindbis virus).

However, the design of a functional chimeric protein for targeting isnot facile. For example, if one wishes to create a chimeric adenoviralknob protein containing an targeting domain, the recombinant knobprotein must be able to (a) assemble properly into the icosahedral viralstructure and (b) also retain the binding specificity of the targetingmoiety. This may involve significant and complex molecular modeling toincorporate the targeting moiety into the appropriate region of the knobprotein to insure that the targeting moiety is on the surface of theknob protein. Additionally, since the precise process for assembly ofthe adenoviral particle is poorly understood it is possible thatinsertion of a large targeting moiety will sufficiently interrupt thethree dimensional structure of the viral protein so that it does notefficiently assemble into an infectious virion. Furthermore, wheneverone wishes to change the targeting properties of the adenovirus, it isnecessary to reengineer the knob protein taking into account all of theforegoing, which can be a lengthy process. Moreover, the manipulation ofthe adenoviral genome to obtain a gene that encodes the chimeric proteinis a time consuming process, due to the size and complexity of theadenoviral genome.

In order to avoid these hurdles, other methods of cell specifictargeting rely on the conjugation of antibodies or antibody fragments tothe envelope proteins (see, e.g. Michael et al. (1993) J. Biol. Chem.268:6866-6869, Watkins et al. (1997) Gene Therapy 4:1004-1012; Douglaset al. (1996) Nature Biotechnology 14: 1574-1578. This approach also hasits limitations. First, in the case of chemically conjugating theantibody (or antibody fragment) to the surface of the virion, thelinkage is generally achieved by modification of amino acyl side chainsin the antibody (particularly through lysine residues). As it isdifficult to control the stoichiometry of this reaction, one canenvision the resulting virion being coated with antibodies in a varietyof orientations. As the binding specificity of the antibody is containedin the variable regions, the random association of the cross-linkedantibody will result in many of the antibody variable domains being“hidden” and thus ineffective. Accordingly, in order to insure asufficient number of exposed variable domains to achieve efficienttargeting, a significant excess of antibody must be complexed to thevirion. Additionally, the coating of the virion with an excess ofantibodies may interfere with internalization of the virus in the targetcell. For example, in the case of adenoviruses, the interaction betweenthe viral coat proteins and the CAR receptor is believed to be anessential step in the infectious process. If the viral coat proteins areobscured by an excess of antibody proteins, one may expect that theefficiency of binding to the CAR receptor and internalization wouldsuffer. If the virion is unable to infect the cell and exert itstherapeutic effect, it is questionable whether this targeting approachwould provide significant therapeutic benefit.

Alternative to the use of antibodies, others have complexed targetingproteins to the surface of the virion. See, e.g. Nilson et al. (1996)Gene Therapy 3:280-286 (conjugation of EGF to retroviral proteins).However, this approach suffers many of the same limitation as the use ofantibodies, such as obscuring viral coat proteins and potentiallyinterfering with the infectious mechanism.

In one attempt to avoid these problems, some groups have used anti-knobor anti-fiber antibodies complexed to a targeting moiety (see, e.g.,U.S. Pat. No. 5,871,727). While this avoids the problem of having aantibody-coated virion as discussed above, such non-covalent complexesare in equilibrium with the free conjugated antibody and virion species,i.e.{conjugated antibody-virion}⇄conjugated-antibody+virion.While the affinity of the antibody for the knob may be high and theresulting equilibrium constant of this reaction suggests the formationof a “stable” complex, this does not indicate that the complex will bekinetically stable in solution over a period of time. Additionally,although a complex may be “stable” in a solution of limited volume, uponintroduction of the complex to a solution of essentially infinite volume(e.g., the bloodstream of a mammal) the equilibrium will be shifted infavor of dissociation of such a complex.

SUMMARY OF THE INVENTION

The present invention provides targeted complexes that are useful fordelivering molecules to a particular cell or tissue type of interest.The invention provides targeted complexes of the formula:{(delivery vehicle-CM)-TMI-(CM-targeting ligand)};

The delivery vehicle can be, for example, a peptide vector, apeptide-DNA aggregate, a liposome, a gas-filled microsome, anencapsulated macromolecule, and the like. In some embodiments, thedelivery vehicle is a viral vector. Particularly suitable viral vectorsinclude a retrovirus, a vaccinia virus, a herpes virus, anadeno-associated virus, a minute virus of mice (MVM), a humanimmunodeficiency virus, a sindbis virus, an MoMLV, and a hepatitisvirus.

“CM” is a chelating moiety, such as a chelating peptide or an organicchelating agent. TMI is a transition metal ion. CM-targeting ligand is achelating moiety (CM) covalently linked to a targeting ligand that canbind to a cell or tissue of interest.

The invention also provides methods for producing a kinetically inerttargeted delivery vehicle complex. These methods involve: a) preparing akinetically labile transition metal complex by contacting a deliveryvehicle-CM and a CM-targeting ligand with a transition metal ion that isin a kinetically labile oxidation state; and b) changing the oxidationstate of the metal ion to form the kinetically inert complex

Also provided by the invention are methods of delivering a therapeuticor diagnostic agent to a target cell in an organism. These methodsinvolve administering to an organism a targeted complex of the formula:{(delivery vehicle-CM)-TMI-(CM-targeting ligand)};

wherein delivery vehicle-CM is a delivery vehicle that displays on itssurface a polypeptide that comprises a chelating moiety (CM), TMI is atransition metal ion, and CM-targeting ligand is a chelating moiety (CM)covalently linked to a targeting ligand that binds to the target cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of one embodiments of the complexes of thepresent invention. The drawing provides is a diagrammatic representationof a. complex wherein the virus is an adenovirus is containing amodified knob domain containing a chelating peptide and the targetingmoiety is a single chain antibody containing a chelating peptidechelating moiety.

FIG. 2 is an enhanced diagrammatic representation of the linkage of thetargeting moiety to the modified viral coat protein. The centralcircular entity represents a transition metal ion. The semi-circularstructure shaded by cross-hatching represents the chelating moiety whichis covalently linked to the targeting moiety. The semi-circularstructure shaded with the dots represents the viral coat protein whichhas been modified to contain a chelating peptide.

DETAILED DESCRIPTION

The present invention provides viral vectors and other delivery vehiclesto which targeting ligands are attached by a kinetically inertcoordinate covalent linkage. The targeting ligands allow the deliveryvehicle to be targeted to a particular cell or tissue type. The viralvectors, for example, display on their virion surface a coat proteinthat has been modified to include a chelating peptide. The targetingligand is attached to a chelating moiety (e.g., a chelating peptide oran organic chelating agent), and a transition metal ion is employed toform a coordinate covalent bond with the modified coat protein and thetargeting ligand. A coordinate covalent bond occurs when a given speciesdonates a lone electron pair to a vacant orbital in another species.

The use of a coordinate covalent bond as a means to attach the targetingligand to the gene delivery system provides significant advantages overpreviously available methods for targeting vectors, which havesignificant limitations as previously discussed. First, one need notreengineer a viral genome, for example, to modify the gene that encodesthe surface protein each time one wishes to use a different targetingligand. One simply employs a different CM-targeting ligand to retargetthe vector and modify its tropism. Second, coordinate covalent complexesare kinetically inert, resulting in a long-lasting targeted vector. Incontrast, attachment of targeting ligands by means of non-covalentlinkage, for example, antibodies that bind to viral coat proteins is notkinetically inert.

It is essential that one appreciate the distinction between akinetically inert and a thermodynamically stable complex. Thisdistinction is discussed in detail in Anderson et al. (U.S. Pat. No.5,439,829 issued Aug. 8, 1995). Thermodynamic stability refers to thethermodynamic tendency of a species to exist under equilibriumconditions. A kinetically inert complex, on the other hand, is one thatis not labile, i.e., a particular complexed ion is not able to readilyengage in reactions that result in replacement of one or more ligands inits coordination sphere by others. For example, in an aqueousenvironment, unoccupied coordination positions on a transition metal ionare occupied by water. A chelating peptide or other chelating agent mustdisplace the water molecules to form a complex. When such reactionsoccur rapidly, the reaction is termed “labile.” However, where suchreactions occur very slowly or not at all, the complex is said to bekinetically “inert.” Kinetic lability or inertness, unlike thermodynamicstability or instability, is thus related to the reaction rate. Acomplex can be thermodynamically stable even though the on/off reactionsoccur very rapidly (see, e.g., Advanced Inorganic Chemistry, Cotton, F.A. and Wilkinson, G. (1972) 3rd ed. Interscience Publishers, p. 652).Conversely, a complex can be kinetically inert, and thus last forperiods of time ranging from days to years, even though the complex isthermodynamically unstable (equilibrium lies in favor of dissociation)because the rate of dissociation is low.

While the affinity of an antibody for a particular protein may be highand the resulting equilibrium constant of this reaction suggests theformation of a “stable” complex, this does not indicate that the complexwill be kinetically stable in solution over a period of time. Thispresents a particularly serious drawback when such non-covalentinteractions are used to attach a targeting ligand to a delivery systemwhich is then introduced into a biological system. The increased volumeupon introduction of the complex to an organism will result in anequilibrium constant (K_(eq)) favoring dissociation, since the bloodvolume is essentially infinitely large in comparison to the administeredvolume. Furthermore, the toxicity of the free components of the complexmay provide an additional degree of uncertainty in the use of suchcomplexes in mammalian systems. Since non-covalently linked complexeswill necessarily result in free species upon administration to anorganism, the toxicity of the free species in addition to the complexwould need to be evaluated. In human beings, this would likelycomplicate the regulatory approval process for such complexes as itwould require additional toxicology clinical studies. These problems areavoided by the present invention, which uses a kinetically inertcoordinate covalent linkage to attach the targeting ligand to the viralcoat protein or other gene delivery system.

I. Targeted Complexes

Generally, the targeted complexes of the invention can be represented bythe formula:{(delivery vehicle-CM)-TMI-(CM-targeting ligand)}  (1)wherein delivery vehicle-CM refers to a delivery vehicle that displayson its surface a chelating moiety, TMI is a transition metal ion, andCM-targeting ligand is a chelating moiety (CM) covalently linked to atargeting ligand. In presently preferred embodiments, the deliveryvehicle is a viral vector, the chelating moiety is a chelating peptide,and the polypeptide to which the chelating peptide is attached is aviral coat protein.

A. Viral Vectors and Other Delivery Vehicles

The present invention provides complexes in which a viral vector orother delivery vehicle is attached by a coordinate covalent linkage to atargeting ligand. Such delivery vehicles include, in addition to viralvectors, other molecules or carriers that are useful for delivering anagent to a cell. Liposomes, for example can be engineered to accept thecoordinate covalently linked targeting ligands, as can molecules thatbind to nucleic acids or other agents.

In some embodiments, the complexes include a viral vector to whichtargeting ligands are attached. The term “virus” is used in itsconventional sense to refer to any of the obligate intracellularparasites having no protein-synthesizing or energy-generating mechanismand generally refers to any of the enveloped or non-enveloped animalviruses commonly employed to deliver exogenous transgenes to mammaliancells. The viruses possess virally encoded viral coat proteins. Theviruses useful in the practice of the present invention includerecombinantly modified enveloped or non-enveloped DNA and RNA viruses.In presently preferred embodiments, the viruses are selected frombaculoviridiae, parvoviridiae, picomoviridiae, herpesviridiae,poxviridae, or adenoviridiae. Chimeric viral vectors which exploitadvantageous elements of each of the parent vector properties (See e.g.,Feng et al. (1997) Nature Biotechnology 15:866-870) can also be employedin the practice of the present invention.

Viral vector systems useful in the practice of the instant inventioninclude, for example, naturally occurring or recombinant viral vectorsystems. For example, viral vectors can be derived from the genome ofhuman or bovine adenoviruses, vaccinia virus, herpes virus,adeno-associated virus (see, e.g., Xiao et al., Brain Res. 756:76-83(1997), minute virus of mice (MVM), HIV, sindbis virus, and retroviruses(including but not limited to Rous sarcoma virus), and MoMLV, hepatitisB virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997).Typically, genes of interest are inserted into such vectors to allowpackaging of the gene construct, typically with accompanying viral DNA,followed by infection of a sensitive host cell and expression of thegene of interest. One example of a preferred recombinant viral vector isthe adenoviral vector delivery system which has a deletion of theprotein IX gene (see, International Patent Application WO 95/11984,which is herein incorporated by reference in its entirety for allpurposes).

In some instances it may be advantageous to use vectors derived from adifferent species from that which is to be treated in order to avoid thepreexisting immune response. For example, equine herpes virus vectorsfor human gene therapy are described in WO98/27216 published Aug. 5,1998. The vectors are described as useful for the treatment of humans asthe equine virus is not pathogenic to humans. Similarly, ovineadenoviral vectors may be used in human gene therapy as they are claimedto avoid the antibodies against the human adenoviral vectors. Suchvectors are described in WO 97/06826 published Apr. 10, 1997.

The virus can be replication competent (e.g., completely wild-type oressentially wild-type such as Ad dl309 or Ad dl520), conditionallyreplicating (designed to replicate under certain conditions) orreplication deficient (substantially incapable of replication in theabsence of a cell line capable of complementing the deleted functions).Alternatively, the viral genome can possess certain modifications to theviral genome to enhance certain desirable properties such as tissueselectivity. For example, deletions in the E1 a region of adenovirusresult in preferential replication and improved replication in tumorcells. The viral genome can also modified to include therapeutictransgenes (as more fully described below). The virus can possesscertain modifications to make it “selectively replicating,” i.e. that itreplicates preferentially in certain cell types or phenotypic cellstates, e.g., cancerous. For example, a tumor or tissue specificpromoter element can be used to drive expression of early viral genesresulting in a virus which preferentially replicates only in certaincell types. Alternatively, one can employ a pathway-selective promoteractive in a normal cell to drive expression of a repressor of viralreplication. For example, a conditionally replicating adenoviral vectorcan be created by the use of a promoter active in the presence ofendogenous p53 to drive expression of the E2F-Rb fusion protein (apotent inhibitor of the E2 adenoviral promoter). In such instances,where there is a defect in the p53 pathway such that active p53 is notpresent (e.g., a tumor cell), the repressor of viral replication is notexpressed and the virus will replicate. However, where p53 is present(e.g. normal cells) the repressor of viral replication is expressed andviral replication is prevented. Selectively replicating adenoviralvectors that replicate preferentially in rapidly dividing cells aredescribed in International Patent Application No. WO1999US0021451 (Publ.No. WO 022136) entitled “Recombinant E1A Deleted Adenoviral Vectors.”These vectors contain modifications to the E1 a coding sequence so as toproduce E1 a gene products that are deficient in binding to one or moreE1 a p300 protein family members and one or more Rb protein familymembers, but retain the transactivating function of the E1 a CR3 domain.Selectively replicating viruses are also described in InternationalPatent Application No. WO1999US0021452 (Publ. No. WO 022137), which isentitled “Selectively Replicating Viral Vectors.” These viral vectorsreplicate in cells that have a defective pathway (e.g., a p53 orTGF-beta pathway), but not in cells with an active pathway.

Additionally, the viral vector may be replication deficient or defectivein that it possesses certain modifications to the viral genome so as toessentially deprive the virus of its ability to replicate in cells thatare not capable to complementing the deleted adenoviral functions. Forexample, recombinant adenoviral vectors possessing a deletion of E1 genefunctions are essentially unable to replicate except in cell lines thathave been engineered to complement E1 functions, such as 293 cells,PERC.6 cells or A549-E1 cells. Such replication defective vectors havebeen used effectively to deliver therapeutic transgenes, such as the p53tumor suppressor gene. Replication defective viral vectors arepreferably derived from adenovirus serotypes 2 or 5 and possessdeletions or mutations in the E1 region rendering one or more earlygenes inoperative so as to attenuate the replication of the virus innon-complementing cells. Additional deletions in the non-essential E3region are also permissible to increase the packaging capacity of suchvectors. Replication defective adenoviral vectors may also containmutations or deletions so as to substantially eliminate protein IXfunction. Particularly preferred adenoviral vectors are described inGregory et al., U.S. Pat. No. 5,932,210, issued Aug. 3, 1999.Alternatively, where large DNA inserts are desired to achieve thetherapeutic effect in the target cell, a “gutted” or minimal viralvector system can be employed. Such vectors are well known in the artand a review of this technology is provided in Morsy and Caskey,Molecular Medicine Today, January 1999 pp. 18-24; Zhang, et al.(WO98/54345A1 published Dec. 3, 1998); and Kochanek et al. (1996) Proc.Nat'l. Acad. Sci. USA 93: 5731-5736.

In a presently preferred embodiment of the invention, the virus is anadenovirus. The use of adenoviral vectors for the delivery of exogenoustransgenes are well known in the art. See, e.g., Zhang, W-W. (1999)Cancer Gene Therapy 6:113-138. The term “adenovirus” refers collectivelyto animal adenoviruses of the genus mastadenovirus including, but notlimited to, human, bovine, ovine, equine, canine, porcine, murine andsimian adenovirus subgenera. In particular, human adenoviruses includethe A-F subgenera as well as the individual serotypes thereof theindividual serotypes and A-F subgenera including but not limited tohuman adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A andAd 11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, and 91. The bovine adenoviruses usefulin the invention include, but are not limited to, bovine adenovirustypes 1,2,3,4,7, and 10. Canine adenoviruses, as used herein, includesbut is not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect,Toronto 26-61) and 2. Equine adenoviruses of interest include, but arenot limited to, equine types 1 and 2 and porcine adenoviruses ofinterest include, for example, porcine types 3 and 4. In a presentlypreferred practice of the invention, the virus is an adenovirus ofserotype 2 or 5.

Adenoviral polypeptides into which one can incorporate a chelatingpeptide include, for example, the fiber protein (see, e.g., U.S. Pat.Nos. 5,846,789, 5,770,442, 5,543,328 and 5,756,086), the penton baseprotein (see, e.g., U.S. Pat. Nos. 5,559,099, 5,731,190 and 5,712,136),and the hexon protein (see, e.g., U.S. Pat. No. 5,922,315).

Retroviral vectors can also be targeted using the coordinate covalentcomplexes of the present invention. The envelope protein of retroviralvectors is modified to include a chelating peptide. The retroviral genethat encodes the env polypeptide is modified so that a fusion between achelating peptide and all or part of the env polypeptide is expressed.Modifications of retroviral env-encoding genes are described in, forexample, U.S. Pat. Nos. 5,869,331. U.S. Pat. No. 5,736,387 describes theuse of chimeric targeting proteins that include a ligand (e.g., acytokine analog) that is capable of binding to a cytokine receptor totarget retroviral vectors to cells that display the cognate cytokinereceptor. Viral vectors having a chimeric envelope protein that binds tocell surface receptors are described in, for example, U.S. Pat. No.5,985,655. The present invention allows such targeting schemes to beaccomplished without having to modify the viral genome for eachdifferent targeting moiety.

Other suitable viral vectors include paramyxovirus, such as simian virus5 (SV5), a common and non-pathogenic RNA virus. Two viral glycoproteinsare found in the envelope of SV5: the HN protein which functions inattachment to host cell receptors, and the F protein which fuses thevirion envelope with the target cell plasma membrane. U.S. Pat. No.5,962,275 describes the engineering of SV5 to encode a foreign proteinin place of the normal viral attachment protein HN. Virions containingthe foreign membrane protein in the viral envelope are specific to cellsexpressing the ligand that is complementary to the virion-associatedforeign protein or glycoprotein. The present invention provides a meansto make such chimeric envelope proteins without having to alter theviral genome each time a different targeting moiety is used. Instead,the viral genome is modified to express at least the virion-boundportion of the HN protein fused to a chelating moiety. No additionalchanges to the viral genome are then required to substitute onetargeting moiety for another. One simply expresses the desired targetingmoiety, linked to a chelating moiety, and attaches it to the genericvirion.

Bacteriophage are another delivery system to which the present inventionis applicable. Targeted bacteriophage vectors are described in, forexample, U.S. Pat. No. 6,054,312.

In some presently preferred embodiments, the viral vector is modified soas to reduce or eliminate the native tropicity of the virus. Forexample, the interaction of the a native viral envelope protein with acell surface receptor is often highly specific and determines cell-typespecificity of a particular virus (Weiss et al. (1985) RNA tumorviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).Therefore, by engineering the chelating peptide so that the portion ofthe env polypeptide that confers cell specificity is disrupted oreliminated, one can obtain a targeted viral complex that is not only hasenhanced affinity for the cell or tissue type that is recognized by thetargeting ligand, but also has reduced or eliminated affinity for thenatural target cell. Similarly, infection of adenoviruses intosusceptible cells involves the binding of the adenovirus fiber protein(in particular, the C-terminal knob domain) to the coxsackievirus andadenovirus receptor (CAR), which serves as the primary cellularreceptor. The subsequent internalization of the virion involvesArg-Gly-Asp (RGD) sequences in the penton base, which interact with thesecondary host cell receptors, integrins α_(v)β3 and α_(v)β₅. Thus, bydisrupting either or both of the fiber protein and the penton base, onecan eliminate the native tropicity of the adenoviral vector (see, e.g.,Douglas et al. (1999) Nature Biotechnology, 17: 470-475; U.S. Pat. No.5,885,808). The disruption of proteins involved in native viral tropismcan be as an intended consequence of the introduction of the chelatingpeptide, or can be accomplished by other manipulations of the viralgenome. Parvoviral vectors are another example of viral vectors that canbe targeted using the modified coat protein-chelating peptide complexedto a targeting ligand.

The invention also provides complexes in which a conformationallyrestrained non-native amino acid sequence is attached to asurface-displayed chelating moiety. Conformationally constrainedpeptides are generally more effective in targeting delivery to specificcells and/or tissues than unconstrained peptides. U.S. Pat. No.6,057,155 describes the use of such conformationally-restrained, or“constrained” amino acid sequences in a chimeric adenovirus fiberprotein. The ability of the chimeric fiber protein to bind to the celland/or mediate cell entry is increased, e.g., relative to the wild-typeprotein. According to U.S. Pat. No. 6,057,155, the conformationalconstraint can be achieved by placing a nonnative amino acid sequence inan exposed loop of the chimeric fiber protein, or, through. theplacement of the sequence in another location and creation of aloop-like structure comprising the nonnative amino acid sequence at thatsite. The present invention facilitates making the chimeric fiberprotein by eliminating the need to alter the viral genome in order tointroduce the nonnative amino acid sequence. Rather, a polypeptide thatincludes a chelating moiety and the nonnative amino acid sequence andassociated loop structure is made by, for example, recombinantexpression. This polypeptide is then attached to a viral vector thatdisplays a corresponding chelating moiety through a transition metalion.

The invention also provides methods for reducing or eliminating theability of a viral vector to be recognized by an antibody that couldotherwise neutralize the vector. Neutralizing antibodies can, forexample, inhibit entry of a vector into a cell, or inhibitvector-mediated gene expression. Therefore, by modifying coat proteinsof the viral vector, one can reduce the susceptibility of the vector toneutralization. U.S. Pat. No. 6,127,525 describes modifying a viral coatprotein to decrease or eliminate the ability of a neutralizing antibodyto interact with an adenoviral vector. These coat protein modificationscan include, for example, introducing non-native amino acids into thecoat protein. For example, a portion of the coat protein amino acidsequence can be removed and replaced with a “spacer” amino acidsequence, or simply by introducing a “spacer” sequence to an unmodifiednaturally occurring coat protein. For example, the deletion of one ormore hypervariable regions (e.g., the I1 loop and/or I2 loops) of theadenoviral hexon protein can result in reduced sensitivity toneutralizing antibodies. Prior to the instant invention, suchmodifications required altering the gene that encodes the respectivecoat protein (e.g., for adenovirus: penton base, hexon, or fiberprotein). Through use of the invention, however, one can simply attachan appropriately modified extracellular region of the coat protein to achelating moiety that is displayed on the surface of the virion using atransition metal ion. Thus, one can readily construct viral vectors thatare appropriate for avoidance of different neutralizing antibodieswithout having to modify the viral genome. A chelating moiety-modifiedextracellular domain molecule is constructed (e.g., by recombinantexpression) for the particular application and attached to the genericviral vector that displays a cell-surface chelating moiety.

Attachment of a targeting moiety by means of a coordinate covalentlinkage according to the invention is useful not only for viral vectors,but also for other delivery vehicles. For example, one can attach atargeting ligand to a liposome using a coordinate covalent linkage. Theliposomes used in these embodiments of the invention carry a chelatingmoiety on their surface. The chelating moiety can be, for example, achelating peptide that is present on a polypeptide that is displayed onthe surface of the liposome membrane. Alternatively chelating peptidesor other chelating moieties can be attached chemically to lipids thatcomprise the liposome membrane. The use of coordinate covalent linkagesfor attaching a targeting ligand to a liposome is advantageous becauseonly one liposome structure need be developed; once such structureshaving chelating moieties are made, it is a simple matter to attach adesired targeting ligand. It is not necessary to reengineer aliposome-anchored polypeptide or other anchoring moiety for each of thetargeting moieties that are of interest.

Coordinate covalent linkages are also useful for attaching targetingmoieties to other vehicles for delivering nucleic acids or othercompounds. For example, one can use these linkages to attach a targetingmoiety to a polycation, which is in turn complexed with a nucleic acidthat is to be targeted to a particular cell or tissue (see, e.g., U.S.Pat. Nos. 5,874,297, 5,166,320, and 5,635,383). For example, geneconstructs or other agents can be conjugated to a cell receptor ligandfor facilitated uptake (e.g., invagination of coated pits andinternalization of the endosome; see, e.g., Wu et al. (1988) J. Biol.Chem. 263:14621-14624; WO 92/06180; U.S. Pat. No. 5,871,727) through acoordinate covalent linkage. Again, the use of coordinate covalentattachment simplifies the attachment of the targeting ligand moleculesto the delivery vehicle.

Other suitable delivery systems include, but are not limited to, an HVJ(Sendai virus)-liposome gene delivery system (see, e.g., Kaneda et al.,Ann. N.Y. Acad. Sci. 811:299-308 (1997)); a “peptide vector” (see, e.g.,Vidal et al., CR Acad. Sci III 32:279-287 (1997)); a peptide-DNAaggregate (see, e.g., Niidome et al., J. Biol. Chem. 272:15307-15312(1997)); lipidic vector systems (see, e.g., Lee et al., Crit Rev TherDrug Carrier Syst. 14:173-206 (1997)); polymer coated liposomes (Marinet al., U.S. Pat. No. 5,213,804, issued May 25, 1993; Woodle et al.,U.S. Pat. No. 5,013,556, issued May 7, 1991); cationic liposomes (Epandet al., U.S. Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A.,U.S. Pat. No. 5,578,475, issued Nov. 26, 1996; Rose et al, U.S. Pat. No.5,279,833, issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No.5,334,761, issued Aug. 2, 1994); gas filled microspheres (Unger et al.,U.S. Pat. No. 5,542,935, issued Aug. 6, 1996), encapsulatedmacromolecules (Low et al. U.S. Pat. No. 5,108,921, issued Apr. 28,1992; Curiel et al., U.S. Pat. No. 5,521,291, issued May 28, 1996;Groman et al., U.S. Pat. No. 5,554,386, issued Sep. 10, 1996; Wu et al.,U.S. Pat. No. 5,166,320, issued Nov. 24, 1992). In each case, thetransition metal ion-mediated chelation methods of the invention can beused to attach a targeting moiety to the delivery vector.

In order to mask the immunogenic effects of the delivery system,especially viral vectors, it may be desirable to additionally complexagents such as polyethylene glycol (PEG) to the surface of the deliverysystem to minimize immunological clearance of the complex. PreferredPEG-ylation protocols are described in Frances et al. (1998) Int. J.Hematology 68:1-18 and commercialized by PolyMASC Pharmaceuticals PLC(London UK) as the “lipoMASC” and “viraMASC” technologies(www.polymasc.com).

B. Chelating Moiety (CM);

The delivery vehicles used in the targeted complexes of the presentinvention include a polypeptide or other molecule that is displayed onthe surface of the delivery vehicle molecule, to which a chelatingmoiety is attached. The term “chelating moiety” (abbreviated herein asCM) refers collectively to chelating peptides and organic chelatingagents. For example, a viral vector can have a coat protein that hasbeen modified to include a chelating peptide. Another chelating moietyis attached to the targeting ligand. The targeting ligand is attached tothe delivery vehicle by means of a transition metal ion that forms acoordinate covalent bond between the CM attached to thesurface-displayed molecule on the delivery vehicle and the CM attachedto the targeting ligand. The CM attached to the delivery vehicle can bethe same as, or different than, the CM that is attached to the targetingligand.

1. Chelating Peptide (CP)

The term “chelating peptide” (abbreviated “CP”) refers to a peptidesequence that is capable of chelating a transition metal ion asdescribed in Smith et al. (U.S. Pat. No. 4,569,794 issued Feb. 11, 1986)and Anderson et al. (U.S. Pat. No. 5,439,829 issued Aug. 8, 1995) theentire teachings of which are herein incorporated by reference.Generally, the chelating peptide is incorporated into the viral coatprotein or other delivery vehicle polypeptide by modifying the viralcoat protein coding sequence. The chelating peptide is incorporated intothe delivery vehicle component at a location that will ensure itsexposure on the delivery vehicle surface. The chelating peptide can beappended to the amino or carboxy terminus of the protein or can beincorporated internally into the delivery vehicle protein in ansurface-exposed domain of the protein.

Examples of an adenovirus in which the knob protein has been modified tocontain a metal chelating peptide are known in the art. For example,Douglas et al. describe a recombinant adenovirus in which a poly-Hismetal chelating peptide has been incorporated into the carboxy terminaldomain of the adenoviral fiber protein (Nature Biotechnology (1999) 17:470-475). The penton and hexon polypeptides are also suitable adenoviruscoat proteins for introduction of the chelating peptide. Apart from theinsertion of the metal chelating peptide in the coat protein, theremainder of the viral genome can be wild-type or can be modifiedthrough conventional recombinant DNA techniques to possess specificproperties.

Chelating peptides that are useful in the targeted vectors of theinvention include, for example, a polyhistidine sequence. Generally, atleast two histidine residues are required to obtain binding to atransition metal ion; the use of additional adjacent histidinesincreases the binding affinity. Typically, six adjacent histidines areused, although one can use more or less than six. Suitable polyhistidinepeptides are described in, for example, Anderson et al. (U.S. Pat. No.5,439,829, issued Aug. 8, 1995), Doebli et al. (U.S. Pat. No. 5,284,993,issued Feb. 8, 1994) and Doebli et al. (U.S. Pat. No. 5,310,663, issuedMay 10, 1994).

In presently preferred embodiments, a nucleotide sequence that encodes achelating peptide is incorporated into a gene that encodes a polypeptidethat is displayed on the surface of a delivery vehicle, and/or thepeptidyl targeting ligand. This typically involves constructing a fusiongene in which a nucleic acid that codes for the polypeptide is linked,in reading frame, to a nucleic acid that codes for the chelatingpeptide. In regard to coat proteins of a virus, the nucleic acidencoding the chelating peptide is preferably placed at a location in thesurface polypeptide gene that does not disrupt the ability of the fusionprotein obtained to be displayed on the surface of the delivery vehicle.Where the targeting ligand is an antibody, the chelatingpeptide-encoding nucleic acid can be placed at or near the region of theantibody gene that encodes the carboxyl terminus of either the lightchain or the heavy chain, or both.

Similarly, when the CP-targeting ligand is created by recombinant means,the nucleotide sequence encoding the chelating peptide is incorporatedinto (or added to) the nucleotide sequence encoding the targetingligand. The chelating peptide should not interfere with the ability ofthe targeting ligand to bind to the target cell or tissue type.

Methods for constructing and expressing genes that encode fusionproteins are well known to those of skill in the art. Examples of thesetechniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, N.Y., (Sambrook et al.); CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion etal., U.S. Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.Alternatively, one can generate CP-targeting ligand species byconventional chemical protein synthesis reactions. For example, anisolated protein can be modified to incorporate a chelating peptide bychemical linkage through the amino or carboxy termini, through freesulfhydryl groups or free E-amino groups of Lysine or Arg.

2. Organic Chelating Agent

The term “organic chelating agents” is used herein to refer non-peptidylbidentate, tridentate, quadridentate, tripod, and macrocyclic ligandscapable of chelating a transition metal ion. Examples of such organicchelators include iminodiacetic acid, nitrilotriacetic acid,terpyridine, bipyridine, triethylenetetraamine, biethylene triamine andderivatives thereof. Suitable chelating moieties are described in, forexample, U.S. Pat. No. 5,439,829.

C. Transition Metal Ion (TMI)

The term “transition metal ion” (abbreviated as TMI), as described inAnderson et al., refers to a variety of metal ions capable of formingcoordinate complex between at least two chelating moieties andpossessing kinetically labile and kinetically inert oxidation states.Octahedral complexes with filled (d⁶) or half-filled(d³) levels such asCr(III), V(II), Mn(IV) and the low spin forms of Co(III), Fe(II),Ru(II), Os(II), Rh(III), Ir(III), Pd(IV), and Pt(IV) tend to beextremely inert and useful in the practice of the instant invention.Hanzik, Robert P. in Inorganic Aspects of Biological and OrganicChemistry, Academic Press, New York, 1976, p. 109. See also, Cotton, F.A. and Wilkinson, G. supra. In the preferred practice of the inventionthe metal ion is selected from the group comprising Te, Co, Cr, and Ru.In the most preferred practice of the invention the metal ion is Co. Inthe most preferred practice of the invention it is desirable to proceedfrom Co(II), Cr(II), or Ru(III) to Co(III), Cr(III), or Ru(II)respectively to form the inert complex. Producing the necessary changein the oxidation state of the metal ion can be achieved by a variety ofredox reagents. For example, oxidizing agents such as oxygen, hydrogenperoxide, and peracids can be used in the practice of the invention.Examples of reducing agents include, for example, thiols, potassiumferrocyanide, potassium thiocyanate, sulfites, and sodium dithionite.These will be prepared in aqueous solutions of appropriateconcentrations.

In some instances, one may wish to incorporate a metal ion which isreadily detected by diagnostic testing equipment such as x-ray ormagnetic resonance imaging. In this manner, a clinician cannon-invasively follow the trafficking of the complex within an organism.Additionally, certain heavy metals such as Te⁹⁹ provide therapeutic(i.e., anti-tumor) effects and can be used to complement the efficacy ofthe vector.

D. Targeting Ligand

The term “targeting ligand” refers to molecules that interact with andbind to cell type surface ligands of particular cells. Examples of suchtargeting moieties include antibodies against cell surface proteins andligands for cell surface proteins. Examples of cell surface proteinsinclude tumor antigens, hormone receptors, G-protein coupled receptors,cytokine receptors, and the like.

1. Antibody

In some embodiments, the targeting ligand includes all or part of anantibody that binds to the desired target tissue or cell. The term“antibody” a term used to collectively describe antibodies, fragments ofantibodies (such as, but not limited to, Fab, Fab′, Fab₂′ and Fvfragments), chimeric, humanized, or CDR-grafted antibodies capable ofbinding antigens of a similar chain polypeptide binding molecules” asdescribed in PCT Application No. PCT/US 87/02208, InternationalPublication No. WO 88/01649, International Publication Date: 10 Mar.1988. Antibodies can be monoclonal or polyclonal, but are preferablymonoclonal. The antibody can be derived from non-human sources (e.g.,mice, rabbits, goats) but when the complexes are being used in thetreatment of human beings, the antibody is preferably a “human” antibodyderived from non-human sources. Transgenic mice have been developedwhich contain the entire human immunoglobulin gene cluster and as suchare capable of producing “human” antibodies. Such technology andservices are available from Abgenix, Inc., 7601 Dumbarton Circle,Fremont, Calif. 94555. As such antibodies are derived from human genes,such antibodies are preferred as targeting ligands due to a reducedpotential immunogenicity to a human host. Again, fragments of such humanantibodies are particularly preferred as targeting ligands. Single chainantibodies modeled on such human antibodies are particularly preferredas they can be prepared more economically in prokaryotic cultureprocedures.

2. Tumor Antigens

When the viral complex is being used to selectively target tumor cells,it is preferred that the targeting ligand is reactive with a tumorantigen. The term “tumor antigen” is used herein to refer to proteinspresent only on tumor cells (tumor specific antigens) as well as thosepresent on normal cells but expressed preferentially on tumor cells(tumor associated antigens). The term tumor antigen includes, but is notlimited to, alfa-fetoprotein (AFP), C-reactive protein (CRP), cancerantigen-50 (CA-50), cancer antigen-125 (CA-125) associated with ovariancancer, cancer antigen 15-3 (CA15-3) associated with breast cancer,cancer antigen-19 (CA-19) and cancer antigen-242 associated withgastrointestinal cancers, carcinoembryonic antigen (CEA), carcinomaassociated antigen (CAA), chromogranin A, epithelial mucin antigen(MC5), human epithelium specific antigen (HEA), Lewis(a)antigen,melanoma antigen, melanoma associated antigens 100, 25, and 150,mucin-like carcinoma-associated antigen, multidrug resistance relatedprotein (MRPm6), multidrug resistance related protein (MRP41), Neuoncogene protein (C-erbB-2), neuron specific enolase (NSE),P-glycoprotein (mdr1 gene product), multidrug-resistance-relatedantigen, p170, multidrug-resistance-related antigen, prostate specificantigen (PSA), CD56, and NCAM. Antibodies which react with such tumorantigens are commercially available or can be prepared throughconventional techniques used for the generation of antibodies.

3. Ligands for Cell Surface Receptors/Proteins

Nearly every cell type in a tissue in a mammalian organism possessessome unique cell surface receptor, e.g., G-protein coupled receptors.Consequently, when targeting delivery of the complex to a particularcell type, it is possible to incorporate nearly any ligand for the cellsurface receptor as a targeting ligand into the complex. For example,peptidyl hormones can be used a targeting moieties to target delivery tothose cells which possess receptors for such hormones. Chemokines andcytokines can similarly be employed as targeting ligands to targetdelivery of the complex to their target cells. A variety of technologieshave been developed to identify genes that are preferentially expressedin certain cells or cell states and one of skill in the art can employsuch technology to identify ligands which are preferentially or uniquelyexpressed on the target tissue of interest. When the ligand is anon-peptidyl or non-protein ligand, it is preferred to employ an organicchelating agent covalently linked to the ligand. When the ligand is aprotein or peptide, it is preferred that the chelating agent is achelating peptide. Again, the chelating peptide can be incorporated atany convenient non-essential domain of the ligand. The preparation ofrecombinant proteins comprising chelating peptides is well known in theart and commercial vectors are available to facilitate the recombinantproduction of proteins incorporating chelating peptides such as thepBlueBacHis2 vector commercially available from Invitrogen, San Diego,Calif.

4. Other Ligands

Other suitable ligands include “totally synthetic affinity reagents,”which are described in U.S. Pat. Nos. 5,948,635, 5,852,167 and5,844,076. Binding polypeptides obtained by directed evolution, forexample, as described in U.S. Pat. No. 5,837,500 can also be used.

Nuclear localization sequences (NLS) can also be attached to a vectorusing transition metal ion chelating methods of the invention. NLSfacilitate trafficking of proteins into a cell nucleus. See, e.g., WO96/41606 and U.S. Pat. No. 6,054,312.

II. Preparing the Targeted Complexes

The invention also provides methods of preparing kinetically inerttransition metal complexes between a chelating peptide that is displayedon a delivery vehicle and a targeting ligand that is attached to achelating moiety. The methods involve:

a) preparing a kinetically labile transition metal complex with atransition metal ion, the delivery vehicle-CM and the CM-targetingligand, and

b) changing the oxidation state of the metal ion to form the kineticallyinert complex.

The formation of the complex while the metal ion is in its kineticallylabile state and then converting the oxidation state to form akinetically inert complex is advantageous the rate of complex formationwith the transition metal ion in its inert state would be very low. Ifit is desired to dissociate the targeting ligand from the deliveryvehicle, this can be accomplished simply by contacting the complex withan appropriate redox reagent to change the oxidation state back to thekinetically labile state.

For embodiments in which the delivery vehicle is a viral vector, themethods of the invention can involve preparing a recombinant viralprotein wherein the viral coat protein possesses a chelating peptide. Arecombinant targeting ligand that is attached to a chelating moiety isalso prepared. The viral coat protein and the targeting ligand are thenreacted with a transition metal ion that is in a kinetically labileoxidation state. To make the complex stable, the oxidation state of thetransition metal ion is changed to a kinetically inert oxidation state.The kinetically inert complexes are then purified.

Each of the species to be complexed (i.e., the CM-delivery vehicle andthe CM-targeting ligand) can be prepared as described above and isolatedusing conventional chromatographic techniques. Preferably, theCM-targeting ligand is purified to homogeneity using CP-IMACpurification as described in Smith et al. (U.S. Pat. No. 4,569,794) andthe CM-virus purified in accordance with the teaching of Shabram et al.(U.S. Pat. No. 5,837,520 issued Nov. 17, 1998, the entire teaching ofwhich is herein incorporated by reference). Alternatively the viralcomplex can be purified using conventional CsC1 procedures.

The formation of a kinetically labile viral complex can be accomplishedby adding the metal ion to the CM-delivery vehicle or the CM-targetingligand independently, or both species can be exposed to the metal ion ina single reaction vessel. However, in order to maximize the yield andavoid the formation of homogenous polymers of delivery vehicle or dimersof targeting ligand-CM species, it is preferred that the metal ion beexposed to the targeting ligand, excess metal removed, and the targetingligand containing the kinetically labile metal be exposed to thedelivery vehicle containing the modified viral coat protein. Adding themetal to, for example, a viral vector first will likely result inpolymerization of the viral particles and precipitation.

The formation of the kinetically inert complex can be achieved using avariety of oxidizing or reducing agents as described above and willdepend on the nature of the metal ion involved. Care should be taken touse any particularly harsh conditions which would result in denaturingof the targeting ligand or CM-delivery vehicles.

The purification of the complexes can be accomplished using conventionalchromatographic techniques. Preferably, the purification/isolation ofthe kinetically inert complexes should be performed in the presence ofimidazole or a similar agent capable of competing with the formation ofa kinetically labile intermediate. This will facilitate the purificationof only kinetically inert complexes by disrupting kinetically labilecomplexes, thus insuring a homogenous kinetically inert complex.

III. Uses of the Targeted Complexes

The complexes of the present invention find use in a wide variety ofapplications. Among these applications are the targeting of therapeuticor diagnostic agents to particular cells or tissues.

A. Therapeutic Applications

The complexes of the present invention are useful in the treatment of awide range of diseases in mammalian organisms. The term “mammalianorganism” includes, but is not limited to, humans, pigs, horses, cattle,dogs, cats, and the like. In these embodiments, a therapeutic agent iscarried in, or attached to, a viral vector, liposome, or other deliveryvehicle, to which is complexed the targeting ligand through a transitionmetal ion.

The methods and compositions of the present invention can be used forthe treatment of a variety of maladies common in mammalian organisms.For example, the formulations and methods of the present invention canbe used for the treatment of a variety of mammalian species sufferingfrom such maladies including humans, pigs, horses, cattle, dogs, cats.In a presently preferred practice of the invention, the mammalianspecies is a human being.

For example, these complexes are useful in the treatment of cancerwherein a viral or other vector targeted to a cancer cell is designed tokill the infected cell, and can be designed to have a by-stander effectso as to kill surrounding cancer cells. In such instances the targetingligand of the complex can be an antibody against a tumor antigen or aligand for a receptor preferentially expressed on target tumor cells. Awide variety of tumor antigens are well known in the art and antibodiesto such antigens are available from commercial sources such as BioDesignInternational (105 York Street, Kennebunk, Me. 04043 USA). Generation ofantibody fragments of such intact antibodies are well known to those ofskill in the art. Additionally, such antibodies can be reengineered tobe chimeric, humanized, etc.

In a particularly preferred embodiment of the invention, the targetingligand is a single chain antibody directed against a tumor antigen suchas CEA. Such single chain antibodies against tumor antigens are known inthe art. For example, Anderson, et al., supra., describe the anti-CEACHEL-13 single chain antibody containing a chelating peptide chelatingmoiety.

1. Gene Delivery

In some embodiments, the complexes of the present invention are used todeliver nucleic acids, including, for example, antisense nucleic acids,genes that encode therapeutic polypeptides, and the like, to specificcells and/or tissues. Nucleic acid delivery is useful for severalapplications, including corrective gene replacement therapy fordefective genes, nucleic acid-mediated immunization, delivery of genesthat encode therapeutic polypeptides, and cancer therapy.

a. Expression systems.

The term “operably linked” refers to a linkage of polynucleotideelements in a functional relationship. A nucleic acid sequence is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the nucleotide sequencesbeing linked are typically contiguous. However, as enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not directly flanked and may evenfunction in trans from a different allele or chromosome.

Expression of a nucleic acid, such as the production of a polypeptide oran antisense nucleic acid, is desired for many applications. Expressionis typically accomplished by placing the nucleic acid to be expressed inan “expression cassette,” which is a nucleic acid construct, generatedrecombinantly or synthetically, that includes nucleic acid elements thatare capable of effecting expression of a structural gene in hostscompatible with such sequences. Expression cassettes include at leastpromoters and optionally, transcription termination signals. Typically,the recombinant expression cassette includes a nucleic acid to betranscribed (e.g., a nucleic acid encoding a desired polypeptide), and apromoter. Additional factors necessary or helpful in effectingexpression may also be used as described herein. For example, anexpression cassette can also include nucleotide sequences that encode asignal sequence that directs secretion of an expressed protein from thehost cell. Transcription termination signals, enhancers, and othernucleic acid sequences that influence gene expression, can also beincluded in an expression cassette.

In order to effect expression of a nucleic acid of interest, the nucleicacid is operably linked to a promoter sequence operable in the mammalcell. Examples of promoters include, for example, viral promotersendogenous to genome of a viral vector, or promoters derived from othersources. The term “promoter” is used in its conventional sense to referto a nucleotide sequence at which the initiation and rate oftranscription of a coding sequence is controlled. The promoter containsthe site at which RNA polymerase binds and also contains sites for thebinding of regulatory factors (such as repressors or transcriptionfactors). Promoters can be naturally occurring or synthetic. Thepromoters can be endogenous to the virus or derived from other sources.The promoter can be constitutively active, or temporally controlled(temporal promoters), activated in response to external stimuli(inducible), active in particular cell type or cell state (selective)constitutive promoters, temporal viral promoters or regulable promoters.

While the complexes of the present invention facilitate targeting toparticular cells, under certain circumstances (particularly where thevirus is designed to destroy the infected cell) it may be desirable tofurther regulate the replication of a replication competent virus orregulate the expression of the nucleic acid. In the preferred practiceof the invention, the promoter is a selective promoter, i.e. promotersthat are preferentially active in selected cell types or cell states.Examples of such selective promoters include tissue specific or tumorspecific promoters. Tissue specific and tumor specific promoters arewell known in the art and include promoters active preferentially insmooth muscle (alpha-actin promoter), epidermal specific (Polakowska etal. U.S. Pat. No. 5,643,746 issued Jul. 1, 1997) pancreas specific(Palmiter et al. (1987) Cell 50:435), liver specific (Rovet et al.(1992) J. Biol. Chem. 267:20765; Lemaigne et al. (1993) J. Biol. Chem.268:19896; Nitsch et al. (1993) Mol. Cell. Biol. 13:4494), stomachspecific (Kovarik et al. (1993) J. Biol. Chem. 268:9917), pituitaryspecific (Rhodes et al. (1993) Genes Dev. 7:913), prostate specific(Henderson, U.S. Pat. No. 5,648,478, issued Jul. 15, 1997), etc. Theterm “selective promoters” also includes promoters which have bothtissue and tumor cell specificity for example the alpha-fetoproteinpromoter is both liver specific and tumor specific replicating much moreefficiently in hepatocellular carcinoma cells than in either non-tumoror non-liver cells.

The term “temporal promoters” refers to promoters which drivetranscription or the therapeutic transgene at a point later in the viralcycle relative to the promoter controlling expression of thepathway-responsive promoter. Examples of such temporally regulatedpromoters include the adenovirus major late promoter (MLP), otherpromoters such as E3. In the preferred practice of the invention, theMLP promoter is employed. In the case of herpes simplex virus genomes,the Latent Activated Promoters is an example of such a temporallyregulated promoter.

The term “inducible promoter” refers to promoters which facilitatetranscription of the therapeutic transgene preferable (or solely) undercertain conditions and/or in response to external chemical or otherstimuli. Examples of inducible promoters are known in the scientificliterature (see, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res.Comm. 230:426-430; Iida et al. (1996) J. Virol. 70(9):6054-6059; Hwanget al. (1997) J. Virol. 71(9):7128-7131; Lee et al. (1997) Mol. Cell.Biol. 17(9):5097-5105; and Dreher et al. (1997) J. Biol. Chem. 272(46);29364-29371. Examples of radiation inducible promoters are described inManome et al. (1998) Human Gene Therapy 9:1409-1417).

b. Therapeutic Transgenes

The term “therapeutic transgene” refers to a nucleotide sequence theexpression of which in the target cell produces a therapeutic effect.The term therapeutic transgene includes but is not limited to tumorsuppressor genes, antigenic genes, cytotoxic genes, cytostatic genes,pro-drug activating genes, apoptotic genes, pharmaceutical genes oranti-angiogenic genes. The vectors of the present invention may be usedto produce one or more therapeutic transgenes, either in tandem throughthe use of IRES elements or through independently regulated promoters.

1) Tumor Suppressor Genes

The term “tumor suppressor gene” refers to a nucleotide sequence, theexpression of which in the target cell is capable of suppressing theneoplastic phenotype and/or inducing apoptosis. Examples of tumorsuppressor genes useful in the practice of the present invention includethe p53 gene, the APC gene, the DPC-4/Smad4 gene, the BRCA-1 gene, theBRCA-2 gene, the WT-1 gene, the retinoblastoma gene (Lee et al. (1987)Nature 329:642), the MMAC-1 gene, the adenomatous polyposis coli protein(Albertsen et al., U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), thedeleted in colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome3p21.3 (Cheng et al. (1998) Proc. Nat'l. Acad. Sci. USA 95:3042-3047),the MTS1 gene, the CDK4 gene, the NF-1 gene, the NF2 gene, and the VHLgene.

2) Antigenic Genes

The term “antigenic genes” refers to a nucleotide sequence, theexpression of which in the target cells results in the production of acell surface antigenic protein capable of recognition by the immunesystem. Examples of antigenic genes include carcinoembryonic antigen(CEA), p53 (as described in Levine, A. PCT International Publication No.WO94/02167 published Feb. 3, 1994). In order to facilitate immunerecognition, the antigenic gene may be fused to the MHC class I antigen.

3) Cytotoxic Genes

The term “cytotoxic gene” refers to nucleotide sequence, the expressionof which in a cell produces a toxic effect. Examples of such cytotoxicgenes include nucleotide sequences encoding Pseudomonas exotoxin, ricintoxin, diphtheria toxin, and the like. Cytotoxic genes are generallyemployed in the situation where the virus is designed to destroy thetargeted cell and as such are particularly preferred in the treatment ofcancer. Given the nature of the toxins produced by such genes, it isdesirable to control the expression of such genes. Consequently, whenthe virus is designed to encode and express a cytotoxic gene, it ispreferred that the promoter be highly selective or able to be closelyregulated.

4) Cytostatic Genes

The term “cytostatic gene” refers to nucleotide sequence, the expressionof which in a cell produces an arrest in the cell cycle. Examples ofsuch cytostatic genes include p21, the retinoblastoma gene, the E2F-Rbfusion protein gene, genes encoding cyclin dependent kinase inhibitorssuch as p16, p15, p18 and p19, the growth arrest specific homeobox (GAX)gene as described in Branellec et al. (PCT Publication WO97/16459published May 9, 1997 and PCT Publication WO96/30385 published Oct. 3,1996). Such genes are generally employed where one does not wish todestroy the targeted cell, but merely to prevent the hyperproliferationof such cells. These genes are particularly useful in the treatment ofbenign hyperproliferative diseases such as glaucoma surgery failure,proliferative vitreoretinopathy. Other ocular diseases associated withexcessive angiogenesis such as age related macular-degeneration,retinopathy of prematurity, and diabetic retinopathy may also be treatedwith such cytostatic genes.

5) Cytokine Genes

The term “cytokine gene” refers to a nucleotide sequence, the expressionof which in a cell produces a cytokine. Examples of such cytokinesinclude GM-CSF, the interleukins, especially IL-1, IL-2, IL-4, IL-12,IL-10, IL-19, IL-20, interferons of the alpha, beta and gamma subtypesespecially interferon α-2b and fusions such as interferon α-2α-1. Inparticular disease states to be treated with cytokines, it is preferredthat the cytokine gene is closely regulated is a dose dependent fashion.For example when using an interferon gene in a vector targeted to livercells, it is preferred that the promoter be able to be closely regulatedby an exogenous substance such as through the use of the GeneSwitch™regulatory system (GeneMedicine, Inc. Woodlands, Tex.).

6) Chemokine Genes

The term “chemokine gene” refers to a nucleotide sequence, theexpression of which in a cell produces a cytokine. The term chemokinerefers to a group of structurally related low-molecular cytokines weightfactors secreted by cells are structurally related having mitogenic,chemotactic or inflammatory activities. They are primarily cationicproteins of 70 to 100 amino acid residues that share four conservedcysteine residues. These proteins can be sorted into two groups based onthe spacing of the two amino-terminal cysteines. In the first group, thetwo cysteines are separated by a single residue (C-x-C), while in thesecond group, they are adjacent (C-C). Examples of member of the ‘C-x-C’chemokines include but are not limited to platelet factor 4 (PF4),platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growthstimulatory activity protein (MGSA), macrophage inflammatory protein 2(MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig alveolarmacrophage chemotactic factors I and I (AMCF-I and -II), pre-B cellgrowth stimulating factor (PBSF),and IP10. Examples of members of the‘C-C’ group include but are not limited to monocyte chemotactic protein1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotacticprotein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophageinflammatory protein 1 α (MIP-1-α), macrophage inflammatory protein 1 β(MIP-1-β), macrophage inflammatory protein 1 γ (MIP-1-γ), macrophageinflammatory protein 3-α (MIP-3-α, macrophage inflammatory protein 3 β(MIP-3-β), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4),macrophage inflammatory protein 5 (MIP-5), LD78 β, RANTES, SIS-epsilon(p500), thymus and activation-regulated chemokine (TARC), eotaxin,I-309, human protein HCC-1/NCC-2, human protein HCC-3, mouse proteinC10.

7) Pharmaceutical Protein Genes

The term “pharmaceutical protein gene” refers to nucleotide sequence,the expression of which results in the production of protein havepharmaceutically effect in the target cell. Examples of suchpharmaceutical genes include the proinsulin gene and analogs (asdescribed in PCT Intemational Patent Application No. WO98/31397, growthhormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH,NGF, VEGF (to increase blood perfusion to target tissue, induceangiogenesis, PCT publication WO98/32859 published Jul. 30, 1998),thrombospondin, etc.

8) Proapoptotic Genes

The term “pro-apoptotic gene” refers to a nucleotide sequence, theexpression thereof results in the programmed cell death of the cell.Such genes are particularly useful in the destruction of the targetedcell for use in cancer therapy. Examples of pro-apoptotic genes includep53, adenovirus E3-11.6K, the adenovirus E4orf4 gene, p53 pathway genes,and genes encoding the caspases.

9) Pro-Drug Activating Genes

The term “pro-drug activating genes” refers to nucleotide sequences, theexpression of which, results in the production of protein capable ofconverting a non-therapeutic compound into a therapeutic compound, whichrenders the cell susceptible to killing by external factors or causes atoxic condition in the cell. An example of a prodrug activating gene isthe cytosine deaminase gene. Cytosine deaminase converts5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a potent antitumoragent. The lysis of the tumor cell provides a localized burst ofcytosine deaminase capable of converting 5FC to 5FU at the localizedpoint of the tumor resulting in the killing of many surrounding tumorcells. This results in the killing of a large number of tumor cellswithout the necessity of infecting these cells with an adenovirus (theso-called bystander effect”). Additionally, the thymidine kinase (TK)gene (see e.g. Woo, et al. U.S. Pat. No. 5,631,236 issued May 20, 1997and Freeman, et al. U.S. Pat. No. 5,601,818 issued Feb. 11, 1997) inwhich the cells expressing the TK gene product are susceptible toselective killing by the administration of gancyclovir can be employed.

10) Anti-Angiogenic and Angiogenesis-Inducing Genes

The term “anti-angiogenic” genes refers to a nucleotide sequence, theexpression of which results in the extracellular secretion ofanti-angiogenic factors. Anti-angiogenesis factors include angiostatin,inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2(as described in Proc. Nat'1. Acad. Sci. USA (1998) 95:8795-8800),endostatin.

Also of interest are angiogenesis-inducing genes that encode, forexample, vascular endothelial growth factor, and other polypeptides thatinduce angiogenesis. Such genes are useful for treating ischemia andother vascular disorders.

It will be readily apparent to those of skill in the art thatmodifications and or deletions to the above referenced genes so as toencode functional subfragments of the wild type protein may be readilyadapted for use in the practice of the present invention. For example,the reference to the p53 gene includes not only the wild type proteinbut also modified p53 proteins. Examples of such modified p53 proteinsinclude modifications to p53 to increase nuclear retention, deletionssuch as the delta13-19 amino acids to eliminate the calpain consensuscleavage site, modifications to the oligomerization domains (asdescribed in Bracco et al. PCT published application WO97/0492 or U.S.Pat. No. 5,573,925).

Furthermore, the above therapeutic genes can be secreted into the mediaor localized to particular intracellular locations by inclusion of atargeting ligand such as a signal peptide or nuclear localization signal(NLS). Also included in the definition of therapeutic transgene arefusion proteins of the therapeutic transgene with the herpes simplexvirus type 1 (HSV-1) structural protein, VP22. Fusion proteinscontaining the VP22 signal, when synthesized in an infected cell, areexported out of the infected cell and efficiently enter surroundingnon-infected cells to a diameter of approximately 16 cells wide. Thissystem is particularly useful in conjunction with transcriptionallyactive proteins (e.g. p53) as the fusion proteins are efficientlytransported to the nuclei of the surrounding cells. See, e.g., Elliott,G. & O'Hare, P. (1997) Cell 88:223-233; Marshall, A. & Castellino, A.(1997) Nature Biotechnology 15:205; O'Hare et al. PCT publicationWO97/05265 published Feb. 13, 1997. A similar NLS derived from the HIVTat protein is also described in Vives et al. (1997) J. Biol. Chem.272:16010-16017.

Additionally, it will be readily apparent to those of skill in the artthat a viral or other vector can be engineered to encode more than onetherapeutic transgene. The transgenes can be the same (for example toincrease the effective gene dosage) or different to achievecomplementary effects. Each transgene can be under control of the samepromoter (for example through the use of IRES elements) or differentpromoters. In those situations where it is desirable to produce a vectorcontaining multiple transgenes, it is preferred to use minimal vectorsystems. The construction of such minimal vectors (also termed “gutted”or “gutless” vectors) are described in Zhang, et al. InternationalPublication No WO9854345A1 and Morsy and Caskey (1999) MolecularMedicine Today, January 1999 issue, pp. 18-24.

2. Other Therapeutic Agents

The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccharides, for example.

B. Diagnostic Applications

The complexes of the invention also find use in diagnostic and labelingapplications. A coordinate covalent linkage mediated by a metal ionjoins a targeting moiety to a detectable label. The label can be presenton a viral or other vector, on a liposome, or can be attached to amolecule that includes a label. Upon administration to an organism, orto a population of cells, the targeting moiety will mediate attachmentof the label to the targeted cells or tissues. One can then detect thepresence of the label to determine which cells and/or tissues have themoiety to which the targeting ligand is directed. Also, as previouslydiscussed, a heavy metal visualizable through conventional diagnosticprocedures can be employed, providing the ability to follow the targetedtherapeutic complex through the organism non-invasively and thusproviding both therapeutic and diagnostic value.

Detectable labels can be primary labels (where the label comprises anelement that is detected directly or that produces a directly detectableelement) or secondary labels (where the detected label binds to aprimary label, as is common in immunological labeling). An introductionto labels, labeling procedures and detection of labels is found in Polakand Van Noorden (1997) Introduction to Immunocytochemistry, 2nd ed.,Springer Verlag, NY and in Haugland (1996) Handbook of FluorescentProbes and Research Chemicals, a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. Primary and secondarylabels can include undetected elements as well as detected elements.Useful primary and secondary labels in the present invention can includespectral labels such as fluorescent dyes (e.g., fluorescein andderivatives such as fluorescein isothiocyanate (FITC) and Oregon Green”,rhodamine and derivatives (e.g., Texas red, tetrarhodimine isothiocynate(TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyes”, andthe like), radiolabels (e.g., 3H, 125I, 35S, 14C, 32P, 33P, etc.),enzymes (e.g., horse radish peroxidase, alkaline phosphatase etc.),spectral colorimetric labels such as colloidal gold or colored glass orplastic (e.g., polystyrene, polypropylene, latex, etc.) beads. The labelmay be coupled directly or indirectly to a component of the detectionassay (e.g., the detection reagent) according to methods well known inthe art. As indicated above, a wide variety of labels may be used, withthe choice of label depending on sensitivity required, ease ofconjugation with the compound, stability requirements, availableinstrumentation, and disposal provisions.

Preferred labels include those that use: 1) chemiluminescence (usinghorseradish peroxidase or luciferase) with substrates that producephotons as breakdown products as described above) with kits beingavailable, e.g., from Molecular Probes, Amersharn, Boehringer-Mannheim,and Life Technologies/Gibco BRL; 2) color production (using bothhorseradish peroxidase and/or alkaline phosphatase with substrates thatproduce a colored precipitate [kits available from LifeTechnologies/Gibco BRL, and Boehringer-Mannheim]); 3) hemifluorescenceusing, e.g., alkaline phosphatase and the substrate AttoPhos [Amersham]or other substrates that produce fluorescent products, 4) fluorescence(e.g., using Cy-5 [Amersharn]), fluorescein, and other fluorescenttags]; 5) radioactivity. Other methods for labeling and detection willbe readily apparent to one skilled in the art.

Preferred enzymes that can be conjugated to targeting ligands using thecoordinate covalent linkages of the invention include, e.g., luciferase,and horse radish peroxidase. The chemiluminescent substrate forluciferase is luciferin. Embodiments of alkaline phosphatase substratesinclude p-nitrophenyl phosphate (pNPP), which is detected with aspectrophotometer; 5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium (BCIP/NBT) and fast red/napthol AS-TR phosphate, which aredetected visually; and 4-methoxy-4-(3-phosphonophenyl)spiro[1,2-dioxetane-3,2′-adamantane], which is detected with aluminometer. Embodiments of horse radish peroxidase substrates include2,2′azino-bis(3-ethylbenzthiazoline-6 sulfonic acid) (ABTS),5-aminosalicylic acid (5AS), o-dianisidine, and o-phenylenediamine(OPD), which are detected with a spectrophotometer; and3,3,5,5′-tetramethylbenzidine (TMB), 3,3′diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art.

In general, a detector which monitors a particular label is used todetect the label. Typical detectors include spectrophotometers,phototubes and photodiodes, microscopes, x-ray, magnetic resonanceimaging (MRI), scintillation counters, cameras, film and the like, aswell as combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

C. Other Uses

The targeted vectors of the invention are also useful to introduce agene into a host for in vivo production of a protein encoded by thegene. For example, transgenic bovines and goats are used for productionof proteins in milk (see, e.g., WO 93/25567). The vectors are alsouseful for making “knockout” animals that are useful for the study ofhuman diseases and other purposes.

IV. Formulations and Treatment Regimes

The complexes prepared above can be formulated for administration to amammalian organism in accordance with techniques well known in the art.The complexes can be administered in conventional solutions such assterile saline and can incorporate one or more carriers of agents topreserve the stability and sterility of the solution. The formulationscan also include carrier molecules conventionally used in theformulation of pharmaceutical agents. The term “carriers” refers tocompounds commonly used on the formulation of pharmaceutical compoundsused to enhance stability, sterility and deliverability of thetherapeutic compound. When the viral, non-viral or protein deliverysystem is formulated as a solution or suspension, the delivery system isin an acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers can be used, e.g., water, buffered water, 0.8% saline,0.3% glycine, hyaluronic acid and the like.

These compositions can be sterilized by conventional, well knownsterilization techniques, or can be sterile filtered. The resultingaqueous solutions can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

The formulations can also include delivery enhancing agents to increaseuptake of the targeted complexes into the target cells. The terms“delivery enhancers” or “delivery enhancing agents” are usedinterchangeably herein and includes agents that facilitate the transferof the nucleic acid or protein molecule to the target cell. Examples ofsuch delivery enhancing agents detergents, alcohols, glycols,surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors,hypertonic salt solutions, and acetates. Suitable alcohols include forexample the aliphatic alcohols such as ethanol, N-propanol, isopropanol,butyl alcohol, acetyl alcohol. Glycols include glycerine,propyleneglycol, polyethyleneglycol and other low molecular weightglycols such as glycerol and thioglycerol. Acetates such as acetic acid,gluconic acid, and sodium acetate are further examples ofdelivery-enhancing agents. Hypertonic salt solutions like 1M NaCl arealso examples of delivery-enhancing agents. Bile salts such astaurocholate, sodium tauro-deoxycholate, deoxycholate,chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid andother astringents such as silver nitrate can be used.Heparin-antagonists like quaternary amines such as protamine sulfate canalso be used. Anionic, cationic, zwitterionic, and nonionic detergentscan also be employed to enhance gene transfer. Exemplary detergentsinclude but are not limited to taurocholate, deoxycholate,taurodeoxycholate, cetylpyridium, benalkonium chloride, Zwittergent 3-14detergent, CHAPS (3-[(3-Cholamidopropyl)dimethylammnoniol]-1-propanesulfonate hydrate), Big CHAP, Deoxy BigCHAP, Triton-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside,PLURONIC-F68 detergent, Tween 20 detergent, and TWEEN 80 detergent(CalBiochem Biochemicals). Particularly preferred delivery enhancingreagents are derivatives of particular impurities that are found in somepreparations of Big CHAP; these derivatives are described in PCTApplication No. US98/14241 (published Jan. 21, 1999 as WO99/02191).

The formulations of the invention are typically administered to enhancetransfer of an agent to a cell. The cell can be provided as part of atissue, such as an epithelial membrane, or as an isolated cell, such asin tissue culture. The cell can be provided in vivo, ex vivo, or invitro. The formulations containing delivery enhancing compounds andmodulating agents can be introduced into the tissue of interest in vivoor ex vivo by a variety of methods. In some embodiments of theinvention, the modulating agent is introduced to cells by such methodsas microinjection, calcium phosphate precipitation, liposome fusion, orbiolistics. In further embodiments, the therapeutic agent is taken updirectly by the tissue of interest.

In some embodiments of the invention, the targeted complexes of theinvention are administered ex vivo to cells or tissues explanted from apatient, then returned to the patient. Examples of ex vivoadministration of therapeutic gene constructs include Arteaga et al.,Cancer Research 56(5): 1098-1103 (1996); Nolta et al. Proc. Nat'l. Acad.Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23(1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996);Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); andMakarov et al., Proc. Nat'l. Acad. Sci. USA 93(1):402-6 (1996).

It will be appreciated by those of skill in the art that the particulardosage of a given complex will depend on a variety of factors. Thetargeted complexes of the present invention provide an advantage overtheir non-targeted counterparts in that a lower dosage can achieve anequivalent therapeutic or diagnostic effect. However, this does notnecessarily mean that a reduced dosage will be indicated in all cases.For example, in oncology applications, administration of the maximumtolerated dose of the therapeutic agent is generally accepted as thepreferred dosage. Clinical trials in human beings have indicated that adose of 2.5×10¹³ adenoviral particles administered for 5 consecutivedays for three courses of therapy is well tolerated (Nielsen et al.(1998) Hum Gene Ther. 9: 681-94). Consequently, viral doses of thismagnitude would be suitable for therapeutic applications. For oncologyapplications the therapeutic agent may also be combined with othertreatment regimens such as radiation, etc.

In non-oncology therapeutic applications and diagnostic applications, amore limited dose would be preferred. Again, the precise nature of thedose will depend on the type of delivery vehicle, the therapeutic ordiagnostic effect sought, the degree of control of transgene, expressionin addition to more common factors such as the patient's age, weight,sex, physical condition, etc. However, the determination of appropriatedose is a matter of routine experimentation to those of skill in theart. Dose escalation trials in mammalian species generally are initiallycarried out in small animal species such as swine, eventually inprimates. Phase I clinical trials in human beings also include such doseescalation and toxicity assessments. Although such experiments aretime-consuming, the skill necessary to achieve the clinically relevantdosage range is a matter of routine experimentation.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A targeted complex of the formula:{(delivery vehicle-CM)-TMI-(CM-targeting ligand)};wherein CM is achelating moiety, TMI is a transition metal ion, and CM-targeting ligandis a chelating moiety (CM) covalently linked to a targeting ligand. 2.The complex of claim 1, wherein the delivery vehicle is a virus and thechelating moiety is a chelating peptide.
 3. The complex of claim 2,wherein the virus lacks a native viral ligand that binds to a nativecellular receptor for the virus.
 4. The complex of claim 2, wherein thevirus is replication competent.
 5. The complex of claim 2, wherein thevirus is replication deficient.
 6. The complex of claim 2, wherein thevirus includes a polynucleotide that encodes a p53 tumor suppressorpolypeptide and the targeting ligand is a antibody that binds to a tumorantigen.
 7. The complex of claim 2, wherein the virus is an adenovirus.8. The complex of claim 7, wherein the viral coat protein is selectedfrom a fiber, a penton and a hexon.
 9. The complex of claim 7, whereinthe adenovirus is replication competent.
 10. The complex of claim 9,wherein the adenovirus is a wild-type adenovirus.
 11. The complex ofclaim 9, wherein the adenovirus is a selectively replicating adenovirus.12. The complex of claim 7, wherein the adenovirus is replicationdeficient.
 13. The complex of claim 12, wherein the genome of theadenovirus comprises a partial or total deletion of the adenoviral E1region.
 14. The complex of claim 12, wherein the genome of theadenovirus comprises a partial or total deletion of the proteinIX-encoding region.
 15. The complex of claim 2, wherein the virus isselected from the group consisting of a retrovirus, a vaccinia virus, aherpes virus, an adeno-associated virus, a minute virus of mice (MVM), ahuman immunodeficiency virus, a sindbis virus, an MoMLV, and a hepatitisvirus.
 16. The complex of claim 1, wherein the delivery vehicle isselected from the group consisting of a bacteriophage, a peptide vector,a peptide-DNA aggregate, a liposome, a gas-filled microsome, and anencapsulated macromolecule.
 17. The complex of claim 1, wherein thetargeting ligand is an antibody.
 18. The complex of claim 17, whereinthe antibody is reactive with a tumor antigen.
 19. The complex of claim17, wherein the antibody is selected from the group consisting of Fab,Fab′, Fab₂′ and Fv fragments.
 20. The complex of claim 17, wherein theantibody is a human antibody.
 21. The complex of claim 17, wherein theantibody is a single chain antibody.
 22. The complex of claim 21,wherein the single chain antibody is reactive with carcinoembryonicantigen.
 23. The complex of claim 1, wherein the targeting ligandcomprises a conformationally constrained peptide.
 24. The complex ofclaim 23, wherein the conformationally constrained peptide comprises aportion of an adenoviral fiber protein.
 25. The complex of claim 1,wherein the CM is a chelating peptide or an organic chelating agent. 26.The complex of claim 25, wherein the organic chelating agent is selectedfrom the group consisting of a bidentate, a tridentate, a quadridentateligand and a tripod ligand.
 27. The complex of claim 26, wherein theorganic chelating agent is selected from the group consisting ofiminodiacetic acid, nitrilotriacetic acid, terpyridine, bipyridine,triethylenetetraamine, and biethylenetriamine.
 28. The complex of claim1, wherein the delivery vehicle is a liposome.
 29. The complex of claim1, wherein the delivery vehicle is a paramyxovirus.
 30. A viral vectorcomplex that comprises a targeting ligand that is attached to a surfacepolypeptide of a viral vector by a coordinate covalent linkage mediatedby a transition metal ion.
 31. A method of producing a kinetically inerttargeted delivery vehicle complex, the method comprising: a) preparing akinetically labile transition metal complex by contacting a deliveryvehicle-CM and a CM-targeting ligand with a transition metal ion that isin a kinetically labile oxidation state; and b) changing the oxidationstate of the metal ion to form the kinetically inert complex.
 32. Themethod of claim 31, wherein the kinetically labile transition metalcomplex is prepared by: a) contacting the CM-targeting ligand with thetransition metal ion in a reaction vessel and allowing the transitionmetal ion to bind to the CM to form a transition metal ion-CM-targetingligand complex; b) removing uncomplexed transition metal ion from thereaction vessel; and c) contacting the transition metal ion-CM-targetingligand complex with the delivery vehicle-CM and allowing the transitionmetal ion to bind to the CM to form the complex.
 33. The method of claim31, wherein the kinetically labile transition metal complex is preparedby contacting the CM-targeting ligand and the delivery vehicle-CM withthe transition metal ion simultaneously.
 34. A method of delivering atherapeutic or diagnostic agent to a target cell in an organism, themethod comprising administering to an organism a targeted complex of theformula:{(delivery vehicle-CM)-TMI-(CM-targeting ligand)};wherein deliveryvehicle-CM is a delivery vehicle that displays on its surface apolypeptide that comprises a chelating moiety (CM), TMI is a transitionmetal ion, and CM-targeting ligand is a chelating moiety (CM) covalentlylinked to a targeting ligand that binds to the target cell.
 35. Themethod of claim 34, wherein the delivery vehicle is a viral vector andthe chelating moiety is a chelating peptide (CP).
 36. The viral vectorof claim 35, wherein the viral vector is selected from the groupconsisting of an adenovirus, a retrovirus, a vaccinia virus, a herpesvirus, an adeno-associated virus, a minute virus of mice (MVM), a humanimmunodeficiency virus, a sindbis virus, an MoMLV, and a hepatitisvirus.
 37. The viral vector of claim 35, wherein the viral vector is anadenoviral vector and the surface polypeptide is a viral coat proteinselected from the group consisting of a penton base, a hexonpolypeptide, and a fiber polypeptide.
 38. The method of claim 34,wherein the therapeutic agent is a gene that encodes a therapeuticpolypeptide.
 39. The method of claim 38, wherein the gene encodes apolypeptide selected from the group consisting of a tumor suppressor, anantigenic polypeptide, a cytotoxic polypeptide, a cytostaticpolypeptide, a cytokine, a chemokine, a pharmaceutical protein, aproapoptotic polypeptide, a prodrug-activating polypeptide, anangiogenesis-inducing polypeptide, and an anti-angiogenic polypeptide.